Glioblastoma (GBM) is a primary brain tumor with a five year survival rate of about five percent. While searching for the cells which initiate GBM tumors, researchers isolated glioblastoma stem cells (GSCs). Implanting a low number of these cells into immunocompromised mice yields a lethal tumor which resembles the pathology of the patient's tumor from which the cells were isolated. Oncolytic herpes simplex viruses (oHSV) selectively kill brain tumor cells while sparing normal brain cells. All oHSVs clinically evaluated so far harbor deletions of the neurovirulence gene ?34.5 for safety. GSCs are not permissive to ?34.5-deficient oHSV replication. In contrast, the non-stem like cell lines isolated from the same GBM tumor as the GSCs are permissive. A suppressor mutation which causes immediate early expression of HSV gene Us11 rescues ?34.5-deficient oHSV replication in GSCs. These observations lead me to hypothesize that GSCs express a unique factor, not expressed in the bulk tumor cells, which blocks ?34.5-deficient oHSV replication. Here I propose using a cell culture system which allows us to observe two different cell populations from the same tumor in order to identify novel factor(s) which inhibit ?34.5-deficient oHSV replication. This proposal seeks to identify GSC restriction factor(s) and characterize the HSV-1 Us11 protein's structural functionality in regards to antagonizing these factor(s).